While currently available antiretroviral therapies are highly effective in suppressing HIV-1 replication in treated patients, these drugs are not curative. Patients are thus required to remain on continuous, lifelong therapy, which has been associated with a variety of toxicities and adverse effects. Also, over time, viral resistance is likely to pose an increasingly serious problem for patients on therapy. The development of novel classes of inhibitors that block steps in the replication cycle distinct from those targeted by currently available drugs is therefore a high priority. We have played a leading role in the development of one such novel class of HIV-1 antiviral drugs, the maturation inhibitors (MIs)._____In the HIV-1 replication cycle, Gag proteins are synthesized as a polyprotein precursor (Pr55Gag) that is cleaved by the viral protease (PR) during virus release from the infected cell. Completion of the Gag processing cascade is essential for virus maturation and infectivity. In collaboration with Panacos Pharmaceuticals, we found that 3-0-(3'-3'-dimethylsuccinyl) betulinic acid (PA-457, now known primarily as bevirimat or BVM) potently inhibits HIV-1 maturation by specifically blocking a late step in the Gag-processing pathway, the conversion of the capsid-spacer peptide 1 (CA-SP1) region of HIV-1 Gag to mature CA. Clinical trials conducted with BVM produced mixed results; in some patients, significant reductions in viral loads were achieved, whereas in other patients, no benefit of BVM therapy was observed. We and others demonstrated that this lack of response was linked to polymorphisms in SP1. We have also studied in detail the target, mechanism of action, and resistance pathways of a second, structurally distinct MI discovered by Pfizer (PF-46396 or PF96). We found that resistance to PF96 was conferred by mutations not only in the vicinity of the CA-SP1 cleavage site [where resistance to BVM maps] but also upstream in the CA major homology region (MHR). Notably, the MHR mutants that arose during selection for PF96 resistance were markedly PF96 dependent. The MHR mutants, which on their own are highly deficient in assembly and replication, could also revert by acquiring a second-site mutation in SP1 residue 8 (T8I). The study of the PF96-dependent MHR mutants and the T8I compensatory mutant is providing a wealth of information about the role of CA and SP1 in assembly and maturation and will advance our understanding of the structural properties of SP1. Our research on BVM and PF96 has provided novel insights into the structure-function relationship between CA and SP1, as well as a framework for increased structural understanding of HIV-1 MI activity._____We have now forged a multidisciplinary collaborative effort, together with chemists in Dr. Joel Schneider's lab (NCI), at DFH Pharmaceuticals, and at the Hetero Research Foundation (HRF), to develop second-generation BVM analogs that are significantly more potent and broadly active against polymorphic isolates of HIV-1 than BVM or PF96. The best of these analogs are undergoing preclinical testing in anticipation of clinical trials. More long term, we hypothesize that defining the structure of the MI-binding pocket in HIV-1 Gag will greatly facilitate the development of novel and more potent inhibitors; to this end, we are collaborating with several structural biology labs to define the structure of the CA-SP1 region in the immature Gag lattice, in both the presence and the absence of bound inhibitor. Our working hypothesis is that MIs block CA-SP1 processing by stabilizing a six-helix bundle that extends from the C-terminus of CA into SP1. Resistance mutations confer escape by stabilizing this helical bundle. We believe that our work in this project will be transformative both in understanding the structure and function of the CA-SP1 region of Gag, which plays critical roles in HIV-1 assembly and maturation, and in developing MIs as a novel class ofantiretroviral agents._____[Corresponds to Freed Project 3 in the July 2016 site visit report of the HIV Dynamics and Replication Program]